1. Field of the Invention
This invention relates to a method for the analysis of technetium present in a mixture as free pertechnetate and bound and unbound, reduced technetium, and more particularly to such a method involving chromatographic techniques. 2. Description of the Prior Art
Radioisotopes have many diagnostic and therapeutic applications in the field of medicine. The various isotopes display differing types and energy levels of radiation, minimum retention times in the body and affinities for body organs and tissue. The selection of an isotope for a particular therapeutic or diagnostic application is dependent upon these varying characteristics.
Technetium, having an atomic number of 43 and an atomic weight of 99, has been used in medical procedures such as for scanning the liver. In preparing the technetium for this type of procedure, it is bound to a carrier such as diphosphonate, polyphosphate, pyrophosphate; iron-ascorbate-DTPA or human serum albumin. In preparing the test solution, however, not all of the technetium will become bound to the carrier. Technetium will primarily assume either a hydrolyzed or reduced state in which the technetium has a charge of +4, or it may assume an oxidized state with a charge of +7. A portion of the reduced technetium will become bound to the carrier, and the remaining reduced technetium will generally be unbound. The oxidized technetium generally is present as free pertechnetate ions having a chemical formula of TcO.sub.4.sup.31. The various chemical states define where the radioactivity will locate in a patient, and it is therefore extremely important to determine the amounts of the various states in the test solution before administering it to a patient.
A method which has been used in the quantitative analysis of a technetium-containing mixture is chromatography. One test involving this technique is the Qualitrol-S system which is marketed by New England Nuclear of North Billerica, Massachusetts. The Qualitrol procedure is designed for use with technetium which is bound to a sulfur colloid carrier. The Qualitrol procedure utilizes the known technique of ascending, thin layer chromatography using silica gel as the support phase or adsorbent, and physiological saline as the developing solvent. A sample of the technetium-containing mixture is spotted on the strip impregnated with silica gel and is air dried. The strip is then placed with one end in contact with the saline solvent and the solvent migrates upwardly. The technetium present as free pertechnetate is transported upwardly with the solvent. The strip is then cut into two pieces and the percentage of free pertechnetate is measured as a ratio of the radioactivity of the top section of the strip to the radioactivity of the entire strip. An estimate of the amount of technetium present as bound technetium is obtained by assuming that all of the technetium is present as either bound technetium or free pertechnetate.
The Qualitrol system does provide a ready means for estimating the amount of bound technetium in a sulfur colloid system. The Qualitrol technique does, however, entail certain drawbacks. The Qualitrol technique is not well suited, for example, for use with a technetium-containing mixture in which there is a possibility of having hydrolyzed reduced technetium. The technetium mixture is dried and developed in air and the presence of reduced technetium will tend to give false positive results for free pertechnetate due to air oxidation. In addition, the accuracy of the estimate as to the amount of bound, reduced technetium becomes correspondingly less accurate as the amount of unbound, reduced technetium present increases. It is also disadvantageous to use the thin layer chromatography support paper which is generally more fragile and therefore more difficult to use than some other support media.
The Seprachrom procedure of the Gelman Instrument Company of Ann Arbor, Michigan, discloses several related chromatography procedures for the analysis of technetium-containing mixtures. The Seprachrom procedures utilize ascending chromatography in conjunction with an instant thin layer chromatography sheet of glass microfiber impregnated with silica gel or polysilicic acid. The technetium-containing mixture is spotted on the support medium and is developed with 85% methanol in most instances, although the same chromatogram is redeveloped with normal saline solution when the carrier is sulfur colloid and with a solvent comprising N-butanol, ethanol and water when the carrier is iron-ascorbate-DTPA. The developing results in the free pertechnetate ions migrating with the solvent to the top of the support medium. The paper is divided and the proportion of the free pertechnetate present is determined by the radioactivity measurements. As in the Qualitrol system, the amount of bound technetium in the technetium-containing mixture is estimated to be the difference between the percentage of free pertechnetate and 100%. The Seprachrom procedure therefore has the disadvantage of failing to provide a fully accurate measurement of the amount of bound, reduced technetium. The Seprachrom procedure also requires a substantial amount of time which may range from between 25 to 60 minutes, and the use of the relatively fragile, instant thin layer chromatography media may also present difficulties.
A third chromatography procedure for the analysis of technetium-containing mixtures has been disclosed by Cooper and Zimmer of the Medical College of Wisconsin, Milwaukee, Wisconsin, in a paper entitled "Radiochemical Purity and Stability of Commercial TC-99m-Stannous DTPA Kits Using a New Chromatography Technique," appearing in the Journal of Nuclear Medicine Technology, Vol. 3, p. 208 (1975). The Zimmer procedure utilizes ascending chromatography on Gelman instant thin layer chromatography strips impregnated with silica gel. A sample of the technetium-containing mixture is spotted on the supporting strip and is fully developed with acetone. The strip is then air dried and redeveloped with normal saline solution. The progress of the migrating saline solution is watched carefully and the development is stopped when the saline solution has reached approximately the center of the developed strip. The strip is then cut into three pieces and the proportionate amount of bound, reduced technetium is determined as the ratio of the radioactivity of the center section to the radioactivity of the entire strip.
The Zimmer procedure therefore provides a direct and accurate measurement of the bound, reduced technetium present in the sample, and may be advantageously used in certain circumstances. The Zimmer procedure does, however, have certain disadvantages when used in other instances. Primarily, the second, partial development with normal saline must be monitored closely to prevent distortion of the test results. If the normal saline is not permitted to migrate the proper extent along the supporting strip, then the radioactivity of the center section will not accurately represent the amount of bound, reduced technetium present in the technetium-containing mixture. The support medium being the instant thin layer chromatography type also presents the handling problems inherent in dealing with a relatively fragile material. Finally, the fact that the same supporting strip must be developed, dried and redeveloped extends the length of time required to complete the analysis. This latter problem is complicated by the fact that the need to divide the paper into three sections generally means that the paper must be longer than for those procedures in which the paper is only cut into two pieces, since the potential for error would otherwise be increased. As a result, the Zimmer procedure is not well suited to such uses as hospital quality control, although it is appropriate for laboratory work.
The development of simultaneous chromatograms is known in the art in the sense that doing more than one thing at a time saves time. Examples of patents disclosing devices designed to facilitate simultaneous performance of two or more chromatographic separations are U.S. Pat. Nos. 3,686,118, issued to Benson on Aug. 22, 1972; U.S. Pat. No. 3,513,092, issued to Matherne on May 19, 1970; U.S. Pat. No. 3,458,437, issued to Ouano on July 29, 1969; an U.S. Pat. No. 3,194,400 issued to Herndon on July 13, 1965. The analysis techniques described above and relating to technetium mixtures have been unable to use the time saving aspect of simultaneous development to any significant advantage.
With the increasing use of radioisotopes, such as technetium, in medicinal applications, and the concommitant need for quality control of such materials, there has arisen a need for a simple and accurate procedure for qualitative analysis of technetium-containing mixtures. The existing techniques have been successfully used for their intended purposes. There remains, however, the desire for an analysis technique which is easy for all persons to perform, and which provides the accuracies required by modern medical applications.
In efficiently and accurately performing chromatographic analyses of this type, it is necessary to permit development of the chromatograms to be complete, while not waiting unnecessarily long after full development to finish the analysis. It is also highly advantageous to know the precise demarcation between the separate zones of the chromatograms for accurate analysis of them. In U.S. Pat. No. 3,046,779, issued to Coleman on July 31, 1962, there is disclosed the use of a ball point pen to draw a line on a paper adsorbent perpendicular to the start line of the solvent. As the solvent migrates longitudinally of the pen line, the ink distorts to indicate the progress of the solvent front. The ink line thereby indicates completion of development, although it does not assist in determining the location of the separated zones of the test sample. An identification system using chromatographic dyes is disclosed in U.S. Pat. No. 3,914,174, issued to Fuchs on Oct. 21, 1975. The adsorbent is coated, preferably by spraying, with two or more different dyestuffs. When an unknown solvent is spotted on the prepared adsorbent, a ring chromatogram characteristic of the particular solvent is formed and the solvent may thereby be identified. The dye does not serve to indicate the completion of the developing or the location of particular zones of the test sample.